DNA methylation is found in most eukaryotic organisms. While the role of differential methylation is still being investigated it is understood that methylation is an important regulatory event in the cell. Methylation is a dynamic process as enzymes are present in cells to methylate as well as demethylate nucleic acids and proteins. Differential methylation of proteins and genes affects activity of proteins and expression of genes. In mammals, gene methylation has been found to be important in X-chromosome inactivation, control of imprinted genes, suppression of testis specific genes and cell type specific expression.
Aberrations in the normal methylated state of genes are correlated to a growing list of diseases or disorders. Diseases associated with aberrant methylation include adult T-cell leukemia, diabetes, various cancers and developmental disorders identified with genetic imprinting, such as Prader-Willi and Angleman's syndrome. In some cases, normally methylated oncogenes become unmethylated allowing their transcription. In other cases, tumor suppressor genes, such as BRCA and RASSF1A, which are normally unmethylated, become methylated resulting in non-suppression of oncogenes. In addition, numerous physiological changes associated with normal aging are now thought to be the effect of changes in the methylation status of genes. In particular, it is thought that the methylation state of CpG islands, those CG-rich areas of genes that are generally found within the promoter region of the gene, are important regulators of gene expression. The methylated state of CpG islands range from fully methylated to hemimethylated to unmethylated. While it is known that methylation plays an important role in gene expression, the mechanisms behind this effect are not fully understood with some genes silenced by methylation while others are expressed.
The methylation state of DNA, RNA and proteins in cells affects cellular signaling through regulation of gene expression and subcellular localization and activation of proteins. DNA methylation is essential for the normal development and functioning of organisms. Aberrant DNA methylation has been linked to developmental diseases and cancer and alterations in DNA and protein methylation and/or acetylation have been documented in studies of age-related neurodegenerative disorders including Alzheimer's disease, Parkinson's disease and Huntington's disease. The most important cofactor in substrate methylation, S-adenosyl-L-methionine (SAM), is administered as a pharmaceutical, under the tradenames Gumbaral and Samyr, either orally or by intramuscular or intraperitoneal injection in Europe and sold over-the-counter, in the United States where it is administered orally to treat disorders such as depression, Parkinson's disease, Alzheimer's disease, dementia, fibromyalgia and schizophrenia.
Protein methylation is a posttranslational modification that regulates biochemical pathways. In these modifications, methyl groups are added to carboxyl groups or side chain nitrogens present in arginine and lysine residues of proteins. Protein methylation plays a role in signal transduction, growth, protein sorting, regulatory mechanisms and the remodeling of chromatin via methylation of histone moieties. Protein methylation is thought to have a role in chemotaxis, insulin secretion and photoreceptor signal transduction. Carboxy methylated proteins include the Ras and Rho families of G-proteins and the protein phosphatase catalytic subunit 2A. N-methylated proteins include cytoskeleton proteins actin and myosin and nuclear proteins; nucleolin, fibrillarin, histones, heterogenous nuclear RNPs and metabolic proteins such as, calmodulin and FGF-2.

S-adenosyl-L-methionine (SAM), formula I, dependent methylation of nucleic acids and proteins plays a crucial role in DNA methylation and the regulation of gene transcription. Some researchers view SAM as the universal methyl donor for both proteins and nucleic acids (Cimato et al., JCB138:1997 1089-1103). The maintenance DNA methyltransferase (DNMT1) utilizes SAM to methylate cytosine at the replication fork to insure fidelity of methylation patterns in normal cells. Flaws in the activity and expression levels of the eukaryotic DNA methyltransferase (MTase) DNMT1 have been integrally linked to oncogenic potential. Thus, non-methylating agents capable of undergoing DNMT1-dependent transfer to DNA might represent an attractive new chemotherapeutic strategy. DNMT1 transfer of non-methyl entities to the methylation sites on DNA may alter transcriptional repression mechanisms controlled through methylation. Such an approach is significantly different from those exemplified by simple inhibition of MTases with SAM analogs. Substances capable of undergoing transfer to nucleic acids in an MTase-dependent way also holds tremendous promise as biochemical tools by which to dissect and understand biological methylation. For instance, posttranslational protein methylation plays a large role in transcription regulation and constitutes an important facet of proteomics. The absence of functionality, however, renders the methyl group difficult to identify and isolate from complex biological mixtures. Thus, substances that take part in SAM-dependent MTase pathways are important proteomic and genomic tools in addition to DNA modifying agents.
Currently, methods used to label polynucleotides for genomic studies rely on the use of DNA polymerases and ligases to incorporate labeled deoxynucleoside triphosphate (dNTPs) bases into the polynucleotide during de novo synthesis. For example, classical sequencing of DNA requires the incorporation of 32P labeled bases by DNA polymerase into the growing DNA strand. More recently, it has become possible to label DNA with fluorophore conjugated dNTPs. Labeling of DNA by these methods is not limited to incorporation of the labeled fragments into the growing DNA molecule by DNA polymerase I but can also be accomplished by end labeling of DNA fragments resulting from a restriction digest. While these methods of labeling polynucleotides using DNA polymerases have revolutionized the ability to sequence and synthesize DNA and RNA, they are limited by the ability of the polymerase to label the DNA in sequential order as the conjugate base is ligated in position with its partner on the template strand.
Present techniques used to determine the methylation state of DNA require complex multi-step procedures. For example, published U.S. patent application 2003/0082609, hereby incorporated by reference in its entirety, describes a method of determining methylation state by chemically pretreating a genomic DNA digest with bisulfite followed by alkaline hydrolysis to convert non-methylated cytosine bases to uracil. The presence of the methylated cytosine is inferred only if it (not a transformed uracil) subsequently hybridizes to a second probe. U.S. Pat. No. 6,617,434, hereby incorporated by reference in its entirety, describes a method of detecting differential methylation at CpG sequences by cutting test and control DNA with a restriction enzyme that will not cut methylated DNA and comparing the two digests. In this instance, the methylated state is inferred by the reduced ability of the enzyme to cut the DNA. Similarly, published U.S. Patent Application 2005/0158731, hereby incorporated by reference in its entirety, describes methods to determine the methylation state of DNA by using a series of restriction enzymes that are alternately methylase sensitive and labeling the ends of the cleaved fragments with 32P followed by gel separation and restriction.

Recently, a 5′-aziridine adenylate cofactor analog as a substitute for SAM was described having the formula II. II can be used in the M.TaqI catalyzed alkylation of adenine within the TaqI recognition sequence d(TCGA). Generally, restriction-modification systems consist of two enzymes, a methylase and a restriction enzyme, each having the same recognition sequences herein denoted by ‘M’ for methylase and ‘R’ for restriction enzyme. In this M.TaqI catalyzed reaction, instead of generating the N6-methyladenine, substrate adenylation is accomplished via ring opening of the aziridine to yield substrate conjugation to the modified cofactor analog. This chemistry is tolerant of cofactor C8 modification and has been successfully used to fluorescently tag short oligonucleotides and large plasmid substrates in an M.TaqI-dependent fashion. The aziridine nucleoside also undergoes M.HhaI-dependent DNA attachment within the M.HhaI recognition sequence d(GCGC) (Pignot, M et al. (1998) Angew. Chem. Int. Ed., 37, 2888-2891; Pljevaljcic, G. et al., (2004) ChemBioChem, 5, 265-269 and U.S. Pat. No. 6,875,750 and published PCT Patent Application WO 00/06587, each hereby incorporated by reference in their entirety).
The 5′ aziridine cofactor analog II, provides a method of attaching a detectable molecule to a methyltransferase substrate using methyltransferase reactions. Thus, physiologically relevant substrates and their sites of methylation can be identified directly, without resorting to a multi-step, indirect process as has been previously necessary. However, one problem to the use of II as a method of methylation detection is that the presence of the modified cofactor analog requires previous linkage to a detectable label such as a fluorophore, as previously described in U.S. Pat. No. 6,875,750. Pre-labeling limits the utility of cofactor analog II, two-fold because such fluorophore linkage may result in steric interference with the substrate and second, prelabeling limits the versatility of use to the pre-determined label.
Published U.S. Patent Applications 2002/0016003, 2003/0199084 and 2005/0148032, each hereby incorporated by reference in its entirety, describe a chemoselective ligation reaction that can be carried out under physiological conditions. This modified Staudinger reaction, the “Staudinger ligation” occurs between an azide and a phosphine in a stable reaction that does not interfere with cell processes to display markers on oligosaccharides present on cell membranes. As disclosed in the above patent applications, the Staudinger ligation, is, generally, directed to cell surface molecules for the purpose of attaching a phosphine-linked tag to the cell membrane.
The further development of compositions and methods to further elucidate the roles of methylation and methylases requires cofactor analogs that are more efficient to construct and have a greater facility of use such that they are compatible with existing technologies and can also be easily modified without affecting their ability to take the place of the native cofactor. For example, the identification of analogs that are easily synthesized and are stable for longer periods of time in storage while also being amenable to labeling with purification moieties and/or reporter moieties would greatly facilitate their use.
These and other features and advantages of various preferred embodiments of the compositions and methods according to this invention are described in, or are apparent from, the following detailed description of various exemplary embodiments of the methods according to this invention.